CFTR modulators like elexacaftor-tezacaftor-ivacaftor (ETI) improve morbidity in cystic fibrosis (CF), but their systemic effects on young children are not yet clear. We hypothesize that ETI has anti-inflammatory effects and reverse disease-associated molecular signatures in children with CF. This exploratory pilot study evaluates the cellular mechanisms of ETI on peripheral immune cells in children (<12 years) with CF using a multi-omics approach. Seventeen children with CF (median age 8.7 years, 26% female) and 12 controls (median age 9.6 years, 42% female) were included for blood RNA-sequencing, proteomics and phosphoproteomics. Baseline pathway enrichment analysis revealed systemic inflammation, transmembrane transporter deficiencies, reduced protein kinase, and GTPase activity. Three months post-ETI, anti-inflammatory effects, epigenetic modulation, and increased protein kinase activity were observed, indicating partial reversal of cellular abnormalities. ETI modified systemic inflammatory, epigenetic, and phosphorylation pathways in young children with CF, offering insights into CF pathology and potential biomarkers for treatment monitoring.

BACKGROUND: Severe bacterial sepsis caused by Klebsiella pneumoniae (KP) is characterized by dysregulated inflammation, multiorgan injury, and high mortality, yet the key molecular drivers of this process remain incompletely understood. This study aimed to elucidate how the Z-DNA binding protein 1 (ZBP1) regulates inflammatory injury during KP-induced sepsis and to identify potential therapeutic targets with translational relevance. METHODS: To investigate the role of macrophage ZBP1 in sepsis, we combined transcriptomic datasets from human sepsis cohorts and murine sepsis and KP-infection models with single-cell RNA sequencing of infected tissues. We further established both global and myeloid-specific Zbp1 knockout (Zbp1 fl/fl Lyz2-Cre +/- ) mice, as well as myeloid-specific Lgmn (encoded Legumain) knockout (Lgmn fl/fl Lyz2-Cre +/- , Lgmn CKO ) mice, to delineate the macrophage-dependent mechanisms of immune regulation. In addition, macrophage-targeted adeno-associated virus 9 (AAV9) vectors driven by the F4/80 promoter were used to overexpress Lgmn in vivo. Flow cytometry, immunofluorescence, and survival analyses were performed to evaluate systemic inflammation, organ injury, and sepsis severity. The effect of recombinant LGMN supplementation on macrophage activation and barrier restoration was also assessed. RESULTS: ZBP1 activation was driven by both type I interferon (IFN-I) signaling and mitochondrial damage. Mechanistically, ZBP1 directly interacted with signal transducer and activator of transcription 3 (STAT3), suppressed its phosphorylation and nuclear translocation, and consequently inhibited transcription of Lgmn, a gene associated with anti-inflammatory macrophage polarization and tissue repair. Genetic and myeloid-specific deletion of Zbp1 resulted in reduced proinflammatory cytokine production and improved survival in both KP- and cecal ligation and puncture (CLP)-induced sepsis models. In contrast, Lgmn CKO aggravated systemic inflammation, organ injury, and barrier dysfunction, whereas macrophage-targeted AAV9-mediated Lgmn overexpression or recombinant LGMN supplementation alleviated macrophage inflammation, restored epithelial function, and improved survival. CONCLUSION: These findings reveal a previously unrecognized ZBP1-STAT3-LGMN signaling axis that contributes to immune and inflammatory dysregulation in KP-induced sepsis and suggest that targeting ZBP1 or restoring LGMN activity may represent a promising therapeutic strategy for severe bacterial infections.

Genomic research on high-consequence Risk Group 4 (RG4) viruses is significantly hindered by the stringent biosafety level 4 (BSL-4) containment requirements, because virus inactivation prior to removal from containment often compromises nucleic acid integrity. As a focused solution to retain nucleic acid integrity as well as safety compliance, we developed and validated a comprehensive suite of optimized workflows compatible with BSL-4 containment and next-generation sequencing applications. We systematically assessed inactivation, homogenization, and RNA extraction methods to establish robust bulk RNA sequencing workflows for diverse sample types. We also adapted state-of-the-art single-cell RNA sequencing (scRNA-seq) by integrating inactivation steps without compromising data quality. To demonstrate feasibility, we conducted scRNA-seq analysis on cells exposed to Lassa virus. Additionally, we optimized viral genome enrichment strategies for high-depth sequencing of multiple RG4 viruses. These validated workflows enable safe, reproducible, and high-resolution genomic analyses of RG4 pathogens, significantly expanding the experimental toolkit for BSL-4 research.